Rat anti perlecan

Cryosectioned cartilage explants were air dried and fixed in neutral-buffered formalin and ice-cold acetone. After treatment with chondroitinase ABC, sections were incubated in blocking buffer (PBS containing 1% goat serum, 5% BSA) and washed in PBS. Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 ​μg/ml) and/or perlecan (rat anti-perlecan, Millipore). After further washing in PBS, sections were incubated with appropriate Alexa Fluor-conjugated secondary antibodies and visualised on an Olympus BX51 fluorescent microscope. Optimal exposure time for each protein was determined as that giving a signal in positively-stained sections and no detectable staining in negatively-stained sections, avoiding over-exposure and signal saturation. Exposure times of individual channels were kept constant. Quantification was done on raw, unaltered images from at least six random regions using ImageJ (NIH, Bethesda, MD).
To validate ADAM15 staining, the anti-ADAM15 antibody was pre-incubated with a 10-fold molar excess of recombinant ADAM15, centrifuged, and the supernatant applied to sections.